This invention relates to a new process for the production of xcex1-human atrial natriuretic polypeptide (hereinafter referred to as the abbreviation xe2x80x9cxcex1-hANPxe2x80x9d) by recombinant DNA technology. More particularly, it relates to a new process for the production of xcex1-hANP by recombinant DNA technology, to chemically synthesized genes for xcex1-hANP and protective peptide-fused xcex1-hANP and to a corresponding recombinant vector and transformant comprising the same.
The xcex1-hANP is a known polypeptide having a diuretic, natriuretic, vasorelaxant and antihypertensive activities Therefore, it may be useful in clinical treatment of hypertension as antihypertensive diuretic agent and has the following structure: 
(Cf. Biochemical and Biophysical Research Communications Vol.118, page 131 (1984)).
The inventors of this invention have newly created a process for the production of xcex1-hANP by recombinant DNA technique using an expression vector comprising a synthetic gene encoding the amino acid sequence (I) of xcex1-hANP. According to this process, xcex1-hANP can be obtained in high yield.
This invention provide a process for the production of xcex1-hANP by (1) culturing a microorganism transformed with an expression vector comprising a synthetic gene encoding an amino acid sequence of a protective peptide-fused xcex1-hANP in a nutrient medium, (2) recovering the protective peptide-fused xcex1-hANP from the cultured broth and (3) removing the protective peptide part of the protective peptide-fused xcex1-hANP.
In the above process, particulars of which are explained in more detail as follows.
The microorganism is a host cell and may include bacteria, fungi, cultured human and animal cells and cultured plant cells. Preferred examples of the microorganism may include bacteria especially a strain belonging to the genus Escherichia (e.g. E. coli HB101 (ATCC 33694), E. coli 294 (ATCC 31446), E. coli (1776) (ATCC 31537), etc).
The expression vector is usually composed of DNA having at least a promoter-operater region, initiation codon, synthetic protective peptide gene, synthetic xcex1-hANP gene, termination codon(s) and replicatable unit.
The promoter-operater region comprises promoter, operater and Shine-Dalgarno(SD) sequence (e.g. AAGG, etc.). The distance between SD sequence and initiation codon is preferably 8-12 b.p. and in the most preferable case as shown in the working Examples mentioned below, the distance between SD sequence and initiation codon (ATG) is 11 b.p. Examples of the promoter-operater region may include conventionally employed promoter-operater region (e.g. lactose-operon, PL-promoter, trp-promoter, etc.) as well as synthetic promoter-operater region. Preferred examples of the promoter-operater region are synthetic trp promoter I, II and III which were newly synthesized by the inventors of this invention and DNA sequences thereof are shown in FIG. 1, 2 and 3, respectively. In the process,there may be used 1-3 consecutive promoter-operater region(s) per expression vector.
Preferred initiation codon may include methionine codon (ATG).
The protective peptide gene may include DNA sequence corresponding to any of peptide or protein which is capable of forming a fused protein with xcex1-hANP and inhibiting undesired degradation of the fused protein in the host cell or the cultured broth. One of preferred examples is xe2x80x9cpeptide Cd genexe2x80x9d linked to xe2x80x9cLH protein genexe2x80x9d (hereinafter xe2x80x9cthe peptide Cd gene linked to LH protein genexe2x80x9d is referred to as xe2x80x9cpeptide CLa genexe2x80x9d), DNA sequence of which is shown in FIG. 4.
The DNA sequence of xcex1-hANP gene is designed from the amino acid sequence of xcex1-hANP, subjected to a number of specific non-obvious criteria. Preferred example of DNA sequence of xcex1-hANP gene is shown in FIG. 5. In the working Examples as mentioned below, between the xcex1-hANP gene and the protective peptide gene, a DNA sequence encoding amino acid lysine is inserted, with the purpose of Achromobacter protease I digestion at the junction of the fused protein.
The termination codon(s) may include conventionally employed termination codon (e.g. TAG, TGA, etc.).
The replicatable unit is a DNA sequence capable of replicating the whole DNA sequence belonging thereto in the host cells and may include natural plasmid, artificially modified plasmid (e.g. DNA fragment prepared from natural plasmid) and synthetic plasmid and preferred examples of the plasmid may include plasmid pBR 322 or artificially modified thereof (DNA fragment obtained from a suitable restriction enzyme treatment of pBR 322). The replicatable unit may contain natural or synthetic terminator (e.g. synthetic fd phage terminator, etc.).
Synthetic preparation of promoter-operater region, initiation codon,protective peptide gene, xcex1-hANP gene and termination codon can be prepared in a conventional manner as generally employed for the preparation of polynucleotides.
The promoter-operater region, initiation codon, protective peptide gene, xcex1-hANP gene and termination codon(s) can consecutively and circularly be linked with an adequate replicatable unit (plasmid) together, if desired using an adequate DNA fragment(s) (e.g. linker, other restriction site , etc.) in a conventional manner (e.g. digestion with restriction enzyme, phosphorylation using T4 polynucleotide kinase, ligation using T4 DNA-ligase) to give an expression vector.
The expression vector can be inserted into a microorganism (host cell). The insertion can be carried out in a conventional manner (e.g. transformation, microinjection, etc.) to give a transformant.
For the production of xcex1-hANP in the process of this invention, thus obtained transformant comprising the expression vector is cultured in a nutrient medium.
The nutrient medium contains carbon source(s) (e.g. glucose, glycerine, mannitol, fructose, lactose, etc.) and inorganic or organic nitrogen source(s) (ammonium sulfate, ammonium chloride, hydrolysate of casein, yeast extract, polypeptone, bactotrypton, beef extracts, etc.). If desired, other nutritious sources (e.g. inorganic salts (e.g. sodium or potassium biphosphate, dipotassium hydrogen phosphate, magnesium chloride, magnesium sulfate, calcium chloride), vitamins (e.g. vitamin B1), antibiotics (e.g. ampicillin), etc.) may be added to the medium.
The culture of transformant may generally be carried out at pH 5.5-8.5 (preferably pH 7-7.5) and 18-40xc2x0 C. (preferably 25-38xc2x0 C.) for 5-50 hours.
Since thus produced protective peptide-fused xcex1-hANP generally exists in cells of the cultured transformant, the cells are collected by filtration or centrifuge, and cell wall and/or cell membrane thereof is destroyed in a conventional manner (e.g. treatment with super sonic waves and/or lysozyme, etc.) to give debris. From the debris, the protective peptide-fused xcex1-hANP can be purified and isolated in a conventional manner as generally employed for the purification and isolation of natural or synthetic proteins (e.g. dissolution of protein with an appropriate solvent (e.g. 8M aqueous urea, 6M guanidine, etc.), dialysis, gel filtration, column chromatography, high performance liquid chromatography, etc.).
The xcex1-hANP can be prepared by cleaving the protective peptide-fused xcex1-hANP in the presence of an appropriate protease (e.g. Achromobacter Protease I (AP I), etc.) treatment or chemical method (e.g. treatment with cyanogen bromide). In the case where C-terminal of the protective peptide is lysine, there can preferably be employed treatment with API. Although API is a known enzyme (Cf. Biochim. Biophys. Acta., 660, 51 (1981)), it has never been reported that fused proteins prepared via recombinant DNA technology can preferably be cleaved by the treatment with API. This method may preferably be employed for cleaving a fused protein composed of peptides having a lysine between a protective peptide and a target peptide having no lysine in its molecule.
The cleavage of the fused protein may be carried out at pH 5-10 and 20-40xc2x0 C. (preferably 35-40xc2x0 C.) for 2-15 hours in an aqueous solution (e.g. buffer solution, aqueous urea, etc.).
In the working Examples as mentioned below, the fused protein is treated with API firstly in a buffer solution containing 8M urea at pH 5, secondly, in a buffer solution containing 4M urea at pH 9. In this condition, the fused protein is cleaved at lysine site, and the produced xcex1-hANP is refolded spontaneously.
Thus produced xcex1-hANP can be purified and isolated from the resultant reaction mixture in a conventional manner as mentioned above.